EP0628078B1 - Multivalente einkettige Antikörper - Google Patents

Multivalente einkettige Antikörper Download PDF

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EP0628078B1
EP0628078B1 EP94903587A EP94903587A EP0628078B1 EP 0628078 B1 EP0628078 B1 EP 0628078B1 EP 94903587 A EP94903587 A EP 94903587A EP 94903587 A EP94903587 A EP 94903587A EP 0628078 B1 EP0628078 B1 EP 0628078B1
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Prior art keywords
single chain
sequence
amino acid
seq
chain antibody
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EP0628078A1 (de
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Peter S. Mezes
Brian B. Gourlie
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Dow Chemical Co
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Dow Chemical Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • C07K16/4266—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig against anti-tumor receptor Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to single chain multivalent antibodies.
  • Antibodies are proteins belonging to a group of immunoglobulins elicited by the immune system in response to a specific antigen or substance which the body deems foreign.
  • the basic structure of an antibody is a tetramer, or a multiple thereof, composed of two identical heterodimers each consisting of a light and a heavy chain.
  • the light chain is composed of one variable (V) and one constant (C) domain, while a heavy chain is composed of one variable and three or more constant domains.
  • the variable domains from both the light and heavy chain designated V L and V H respectively, determine the specificity of an immunoglobulin, while the constant (C) domains carry out various effector functions.
  • variable domain comprises three complementarity determining regions (CDR) flanked by four relatively conserved framework regions (FR).
  • CDR complementarity determining regions
  • FR relatively conserved framework regions
  • the CDR have been assumed to be responsible for the binding specificity of individual antibodies and to account for the diversity of binding of antibodies.
  • antibodies are multivalent molecules.
  • the IgG classes have two identical antigen binding sites, while the pentameric IgM class has 10 identical binding sites.
  • Monoclonal antibodies having identical genetic parentage and binding specificity have been useful both as diagnostic and therapeutic agents.
  • Monoclonal antibodies are routinely produced by hybridomas generated by fusion of mouse lymphoid cells with an appropriate mouse myeloma cell line according to established procedures.
  • the administration of murine antibodies for in vivo therapy and diagnostics in humans is limited however, due to the human anti-mouse antibody response illicited by the human immune system.
  • Chimeric antibodies in which the binding or variable regions of antibodies derived from one species are combined with the constant regions of antibodies derived from a different species, have been produced by recombinant DNA methodology. See, for example, Sahagen et al., J. Immunol., 137 : 1066-1074(1986); Sun et al., Proc . Natl. Acad. Sci. USA, 82 : 214-218 (1987) ; Nishimura et al., Cancer Res., 47 :999-1005 (1987) ; and Lie et al. Proc Natl. Acad. Sci. USA, 84 : 3439-3443 (1987) which disclose chimeric antibodies to tumor-associated antigens.
  • the variable region of a murine anti body is joined with the constant region of a human antibody. It is expected that as such chimeric antibodies are largely human in composition, they will be substantially less immunogenic than murine antibodies.
  • Chimeric antibodies still carry the Fc regions which are not necessary for antigen binding, but constitute a major portion of the overall antibody structure which affects its pharmacokinetics.
  • antibody-like molecules which localize and bind to the target tissue rapidly and for the unbound material to quickly clear from the body.
  • smaller antibody fragments have greater capillary permeability and are more rapidly cleared from the body than whole antibodies.
  • the scFvs have one binding site as compared to the minimum of two for complete antibodies, the scFvs have reduced avidity as compared to the antibody containing two or more binding sites.
  • scFvs having more than one binding site to enhance the avidity of the polypeptide, and retain or increase their antigen recognition properties.
  • multivalent scFvs which are bispecific to allow for recognition of different epitopes on the target tissue, to allow for antibody-based recruitment of other immune effector functions, or allow antibody capture of a therapeutic or diagnostic moiety.
  • the present invention is a multivalent single chain antibody having affinity for an antigen wherein the multivalent single chain antibody comprises two or more light chain variable domains and two or more heavy chain variable domains; wherein, each variable domain is linked to at least one other variable domain.
  • the present invention is a multivalent single chain antibody which comprises two or more single chain antibody fragments, each single chain antibody fragment specifically binding an antigen, wherein the single chain antibody fragments are covalently linked by a first peptide linker which contains an amino acid sequence of and each single chain antibody fragment comprises
  • the invention provides a DNA sequence which codes for a multivalent single chain antibody, the multivalent single chain antibody comprising two or more single chain antibody fragments, each fragment having affinity for an antigen, wherein the fragments are covalently linked by a first peptide linker which contains an amino acid sequence of and each fragment comprising
  • the multivalent single chain antibodies allow for the construction of an antibody fragment which has the specificity and avidity of a whole antibody but are smaller in size allowing for more rapid capillary permeability. Multivalent single chain antibodies also allow for the construction of a multivalent single chain antibody wherein the binding sites can be two different antigenic determinants.
  • Figure 1 illustrates covalently linked single chain antibodies having the configuration V L -L-V H -L-V L -L-V H (LHLH) and V L -L-V H -L-V H -L-V L (LHHL) and a noncovalently linked Fv single chain antibody (Fv2).
  • Figure 2 illustrates the nucleotide sequence of CC49 V L (SEQ ID NO: 1).
  • Figure 3 illustrates the amino acid sequence of CC49 V L (SEQ ID NO: 2).
  • Figure 4 illustrates the nucleotide sequence of CC49 V H (SEQ ID NO: 3) .
  • Figure 5 illustrates the amino acid sequence of CC49 V H (SEQ ID NO:4).
  • Figure 6 illustrates the nucleotide sequence and amino acid sequence of the CC49 single chain antibody LHLH in p49LHLH (SEQ ID NO:6).
  • Figure 7 illustrates the nucleotide sequence and amino acid sequence of the CC49 single antibody LHHL in p49LHHL (SEQ ID NO:8).
  • Figure 8 illustrates construction of plasmids pSL301 T and pSL301 HT.
  • Figure 9 illustrates construction of plasmid p49LHHL.
  • Figure 10 illustrates construction of plasmid p49LHLH.
  • Figure 11 illustrates the results of a competition assay using CC49 IgG, CC49 scFv2, and CC49 scFv using biotinylated CC49 IgG as competitor.
  • single chain antibody fragment or "antibody fragment” as used herein means a polypeptide containing a V L domain linked to a V H domain by a peptide linker (L), represented by V L -L-V H .
  • L peptide linker
  • the order of the V L and V H domains can be reversed to obtain polypeptides represented as V H -L-V L .
  • Domain is a segment of protein that assumes a discrete function, such as antigen binding or antigen recognition.
  • a “multivalent single chain antibody” means two or more single chain antibody fragments covalently linked by a peptide linker.
  • the antibody fragments can be joined to form bivalent single chain anti bodies having the order of the V L and V H domains as follows: V L -L-V H -L-V L -L-V H ; V L -L-V H -L-V H -L-V L ; V H -L-V L -L-V H -L-V L ; or V H -L-V L -L-V L -L-V H .
  • Single chain multivalent antibodies which are trivalent and greater have one or more anti body fragments joined to a bivalent single chain antibody by an additional interpeptide linker.
  • the number of V L and V H domains is equivalent.
  • the present invention also provides for multivalent single chain antibodies which can be designated V H -L-V H -L-V L -L-V L or V L -L-V L -L-V H -L-V H .
  • Covalently linked single chain antibodies having the configuration V L -L-V H -L-V L -L--V H (LHLH) and V L -L-V H -L-V H -L-V L (LHHL) are illustrated in Figure 1.
  • a noncovalently linked Fv single chain antibody (Fv2) is also illustrated in Figure 1.
  • the single chain antibody fragments for use in the present invention can be derived from the light and/or heavy chain variable domains of any antibody.
  • the light and heavy chain variable domains are specific for the same antigen.
  • the individual antibody fragments which are joined to form a multivalent single chain antibody may be directed against the same antigen or can be directed against different antigens.
  • a source of the genes encoding for these regions is required.
  • the appropriate DNA sequence can be obtained from published sources or can be obtained by standard procedures known in the art. For example, Kabat et al., Sequences of Proteins of Immunological Interest 4th ed., (1991), published by The U.S. Department of Health and Human Services, discloses sequences of most of the anti body variable regions which have been described to date.
  • c DNA sequences obtained from mRNA by reverse transcriptase mediated synthesis as a source of DNA to clone into a vector.
  • the source of mRNA can be obtained from a wide range of hybridomas. See, for example, the catalogue ATCC Cell Lines and Hybridomas, American Type Culture Collection, 20309 Parklawn Drive, Rockville Md., USA (1990). Hybridomas secreting monoclonal antibodies reactive with a wide variety of antigens are listed therein, are available from the collection, and usable in the present invention. These cell lines and others of similar nature can be utilized as a source of mRNA coding for the variable domains or to obtain antibody protein to determine amino acid sequence of the monoclonal antibody itself.
  • Variable regions of antibodies can also be derived by immunizing an appropriate vertebrate, normally a domestic animal, and most conveniently a mouse.
  • the immunogen will be the antigen of interest, or where a hapten, an antigenic conjugate of the hapten to an antigen such as keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • the immunization may be carried out conventionally with one or more repeated injections of the immunogen into the host mammal, normally at two to three week intervals. Usually, three days after the last challenge, the spleen is removed and dissociated into single cells to be used for cell fusion to provide hybridomas from which mRNA can readily be obtained by standard procedures known in the art.
  • the V L and V H domains for use in the present invention are preferably obtained from one of a series of CC antibodies against tumor-associated glycoprotein 72 antigen (TAG-72) disclosed in published PCT Applicadon WO 90/04410 on May 3, 1990, and published PCT Application WO 89/00692 on January 26, 1989. More preferred are the V L and V H domains from the monoclonal antibody designated CC49 in PCT Publications WO 90/04410 and WO 89/00692.
  • the nucleotide sequence (SEQ ID NO: 1) which codes for the V L of CC49 is substantially the same as that given in Figure 2.
  • the amino acid sequence (SEQ ID NO: 2) of the V L of CC49 is substantially the same as that given in Figure 3.
  • the nucleotide sequence (SEQ ID NO: 3) which codes for the V H of CC49 is substantially the same as that given in Figure 4.
  • the amino acid sequence (SEQ ID NO: 4) for the V H of CC49 is substantially the same as that given in Figure 5.
  • Suitable linkers for joining the V H and V L domains are those which allow the V H and V L domains to fold into a single polypeptide chain which will have a three dimensional structure very similar to the original structure of a whole antibody and thus maintain the binding specificity of the whole antibody from which antibody fragment is derived.
  • Suitable linkers for linking the scFvs are those which allow the linking of two or more scFvs such that the V H and V L domains of each immunoglobulin fragment have a three dimensional structure such that each fragment maintains the binding specificity of the whole antibody from which the immunoglobulin fragment is derived.
  • Linkers having the desired properties can be obtained by the method disclosed in U.S. Patent 4,946,778, the disclosure of which is hereby incorporated by reference. From the polypeptide sequences generated by the methods described in the 4,946,778, genetic sequences coding for the polypeptide can be obtained.
  • the peptide linker joining the V H and V L domains to form a scFv and the peptide linker joining two or more scFvs to form a multivalent single chain antibody have substantially the same amino acid sequence.
  • linker peptides be attached to the antibody fragments such that the binding of the linker to the individual antibody fragments does not interfere with the binding capacity of the antigen recognition site.
  • a preferred linker is based on the helical linker designated 205C as disclosed in Pantoliano et al. Biochem., 30 10117-10125 (1991) but with the first and last amino acids changed because of the codon dictated by the Xho I site at one end and the Hind III site at the other.
  • the amino acid sequence (SEQ ID NO: 5) of the preferred linker is as follows:
  • the linker is generally 10 to 50 amino acid residues. Preferably, the linker is 10 to 30 amino acid residues. More preferably the linker is 12 to 30 amino acid residues. Most preferred is a linker of 15 to 25 amino acid residues.
  • Expression vehicles for production of the molecules of the invention include plasmids or other vectors.
  • such vectors contain replicon and control sequences which are derived from species compatible with a host cell.
  • the vector ordinarily carries a replicon site, as well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • E. coli is readily transformed using pBR322 [Bolivar et al., Gene, 2 , 95-(1977), or Sambrook et al., Molecular Cloning, Cold Spring Harbor Press, New York, 2nd Ed. (1989)].
  • Plasmids suitable for eukaryotic cells may also be used.
  • S. cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains, such as Pichia pastoris, are available.
  • Cultures of cells derived from multicellular organisms such as SP2/0 or Chinese Hamster Ovary (CHO), which are available from the ATCC, may also be used as hosts.
  • Typical of vector plasmids suitable for mammalian cells are pSV2neo and pSV2gpt (ATCC); pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d (International Biotechnology, Inc.).
  • prokaryotic and eukaryotic viral expression vectors to express the genes for polypeptides of the present invention is also contemplated.
  • the expression vectors and the inserts which code for the single chain multivalent antibodies have compatible restriction sites at the insertion junctions and that those restriction sites are unique to the areas of insertion. Both vector and insert are treated with restriction endonucleases and then ligated by any of a variety of methods such as those described in Sambrook et al., supra.
  • Preferred genetic constructions of vectors for production of single chain multivalent antibodies of the present invention are those which contain a constitutively active transcriptional promoter, a region encoding signal peptide which will direct synthesis/secretion of the nascent single chain polypeptide out of the cell.
  • the expression rate is commensurate with the transport, folding and assembly steps to avoid accumulation of the polypeptide as insoluble material.
  • additional elements may also be needed for optimal synthesis of single chain polypeptide. These elements may include splice signals, as well as transcription promoter, enhancers, and termination signals.
  • additional genes and their products may be required to facilitate assembly and folding (chaperones).
  • Vectors which are commercially available can easily be altered to meet the above criteria for a vector. Such alterations are easily performed by those of ordinary skill in the art in light of the available literature and the teachings herein.
  • the cloning vector contain a selectable marker, such as a drug resistance marker or other marker which causes expression of a selectable trait by the host cell.
  • a selectable marker such as a drug resistance marker or other marker which causes expression of a selectable trait by the host cell.
  • "Host cell” refers to cells which can be recombinantly transformed with vectors constructed using recombinant DNA techniques. A drug resistance or other selectable marker is intended in part to facilitate in the selection of transformants. Additionally, the presence of a selectable marker, such as a drug resistance marker, may be of use in keeping contaminating microorganisms from multiplying in the culture medium. In this embodiment, such a pure culture of the transformed host cell would be obtained by culturing the cells under conditions which require the induced phenotype for survival.
  • Recovery and purification of the present invention can be accomplished using standard techniques known in the art. For example, if they are secreted into the culture medium, the single chain multivalent antibodies can be concentrated by ultrafiltration. When the polypeptides are transported to the periplasmic space of a host cell, purification can be accomplished by osmotically shocking the cells, and proceeding with ultrafiltration, antigen affinity column chromatography or column chromatography using ion exchange chromatography and gel filtration. Polypeptides which are insoluble and present as refractile bodies, also called inclusion bodies, can be purified by lysis of the cells, repeated centrifugation and washing to isolate the inclusion bodies, solubilization, such as with guanidine-HCI, and refolding followed by purification of the biologically active molecules.
  • the activity of single chain multivalent antibodies can be measured by standard assays known in the art, for example competition assays, enzyme-linked immunosorbant assay (ELISA), and radioimmunoassay (RIA).
  • competition assays enzyme-linked immunosorbant assay (ELISA)
  • RIA radioimmunoassay
  • the multivalent single chain antibodies of the present invention provide unique benefits for use in diagnostics and therapeutics.
  • the use of multivalent single chain antibodies afford a number of advantages over the use of larger fragments or entire antibody molecules. They reach their target tissue more rapidly, and are cleared more quickly from the body.
  • the multivalent single chain antibodies can be constructed such that one or more anti body fragments are directed against a target tissue and one or more anti body fragments are directed against a diagnostic or therapeutic agent.
  • the invention also concerns pharmaceutical compositions which are particularly advantageous for use in the diagnosis and/or therapy of diseases, such as cancer, where target antigens are often expressed on the surface of cells.
  • diseases such as cancer
  • target antigens are often expressed on the surface of cells.
  • the multivalent single chain antibodies can be conjugated with an appropriate imaging or therapeutic agent by methods known in the art.
  • the pharmaceutical compositions of the invention are prepared by methods known in the art, e.g., by conventional mixing, dissolving or lyophilizing processes.
  • CC49 A murine monoclonal antibody specific to the human tumor-associated glycoprotein 72 (TAG-72) deposited as ATCC No. HB9459.
  • CC49 FAB An antigen binding portion of CC49 consisting of an intact light chain linked to the N-terminal portion of the heavy chain.
  • CC49 scFv Single chain antibody fragment consisting of two variable domains of CC49 antibody joined by a peptide linker.
  • CC49 Fv2 Two CC49 scFv non-covalently linked to form a dimer.
  • the number after Fv refers to the number of monomer subunits of a given molecule, e.g., CC49 Fv6 refers to the hexamer multimers.
  • CC49 scFv2 Covalently-linked single chain antibody fragment consisting of two CC49 V L domains and two VH domains joined by three linkers.
  • Six possible combinations for the order of linking the V L (L) and the V H (H) domains together are: LHLH, LHHL, LLHH, HLLH, HLHL, and HHLL.
  • pSCFV UHM Plasmid containing coding sequence for scFv consisting of a CC49 variable light chain and a CC49 variable heavy chain joined by a 25 amino acid linker.
  • p49LHLH or p49LHHL Plasmids containing the coding sequence for producing CC49 scFv2 LHLH or LHHL products, respectively.
  • oligonuclotides were synthesized on either a Model 380A or a Model 391 DNA Synthesizer from Applied Biosystems (Foster City, CA) using standard ⁇ -cyanoethyl phosphoramidites and synthesis columns. Protecting groups on the product were removed by heating in concentrated ammonium hydroxide at 55°C for 6 to 15 hours. The ammonium hydroxide was removed through evaporation and the crude mixtures were resuspended in 30 to 40 ⁇ L of sterile water. After electrophoresis on polyacrylamide-urea gels, the oligos were visualized using short wavelength ultraviolet (UV) light.
  • UV ultraviolet
  • DNA bands were excised from the gel and eluted into 1 mL of 100 mM Tris-HCl, pH 7.4, 500 mM NaCl, 5 mM EDTA over 2 hours at 65°C. Final purification was achieved by applying the DNA to Sep-PacTM C-18 columns (Millipore, Bedford, MA) and eluting the bound oligos with 60 percent methanol. The solution volume was reduced to approximately 50 ⁇ L and the DNA concentration was determined by measuring the optical density at 260 nm (OD 260 ).
  • the gel slices were placed In dialysis tubing (Union Carbide Corp., Chicago) containing 5 mM Tris, 2.5 mM acetic acid, 1 mM EDTA, pH 8.0 and eluted using a Max Submarine electrophoresis apparatus (Hoefer Scientific Instruments, CA). Sample volumes were reduced on a Speed Vac Concentrator (Savant Instruments, Inc., NY). The DNA was ethanol precipitated and redissolved in sterile water.
  • TAG-72 antigen prepared substantially as described by Johnson et al, Can. Res., 46 , 850-857 (1986), was adsorbed onto the wells of a polyvinyl chloride 96 well microtiter plate (Dynatech Laboratories, Inc., Chantilly, VA) by drying overnight. The plate was blocked with 1 percent BSA in PBS for 1 hour at 31°C and then washed 3 times with 200 ⁇ L of PBS, 0.05 percent Tween-20. 25 ⁇ L of test antibodies and 25 ⁇ L of biotinylated CC49 (1/20,000 dilution of a 1 mg/mL solution) were added to the wells and the plate incubated for 30 minutes at 31°C.
  • TAG-72 bound to the plate was determined empirically in order not to have excess of either antigen or biotinylated CC49, yet have enough signal to detect competition by scFv.
  • Positive controls were CC49 at 5 ⁇ g/mL and CC49 Fab at 10 ⁇ g/mL.
  • Negative controls were 1 percent BSA in PBS and/or concentrated LB. Unbound proteins were washed away.
  • Samples for SDS-PAGE analysis (20 ⁇ L) were prepared by boiling in a non-reducing sample preparation buffer-Seprasol I (Integrated Separation Systems (ISS), Natick, MA) for 5 minutes and loaded on 10-20 percent gradient polyacrylamide Daiichi Minigels as per the manufacturer's directions (ISS).
  • ISS Integrated Separation Systems
  • Electrophoresis was conducted using a Mini 2-gel apparatus (ISS) at 55 mA per gel at constant current for approximately 75 minutes. Gels were stained in Coomassie Brilliant Blue R-250 (Bio-Rad, Richmond, CA) for at least 1 hour and destained. Molecular weight standards were prestained (Mid Range Kit, Diversified Biotech, Newton Center, MA) and included the following proteins: Phosphorylase b, glutamate dehydrogenase, ovalbumin, lactate dehydrogenase, carbonic amhydrase, B-lactoglobulin and cytochrome C. The corresponding MWs are: 95,500, 55,000, 43,000, 36,000, 29,000, 18,400, and 12,400, respectively.
  • a sheet of Whatman 3MM filter paper was soaked in anode buffer #1 and smoothly placed on the electrode surface.
  • Another filter paper soaked in anode buffer #2 (25 mM tris pH 10.4) was placed on top of the first one.
  • a sandwich was made by next adding the wetted PVDF membrane, placing the equilibrated gel on top of this and finally adding a sheet of filter paper soaked in cathode buffer (25mM Tris-HCI, pH 9.4 in 40 mM glycine). Transfer was accomplished in 30 minutes using 250 mA constant current (initial voltage ranged from 8-20 volts).
  • TBS Tris-buffered saline
  • the probe antibody used was 20 m L bioti nylated FAID14 solution (10 ⁇ g per 20 mL antibody buffer).
  • Antibody buffer was made by adding 1 g BSA per 100 mL of TTBS. After probing for 30-60 minutes at ambient temperature, the membrane was washed 3 times with TTBS, as above.
  • the membrane was incubated for 30-60 minutes at ambient temperature with 20 mL of a 1:500 dilution in antibody buffer of streptavidin conjugated with alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). The wash step was again repeated after this, as above. Prior to the color reaction, membranes were washed for 2 minutes in an alkaline carbonate buffer (20 mL). This buffer is 0.1 M sodium bicarbonate, 1 mM MgCl 2 ⁇ H 2 O, pH 9.8. To make up the substrate for alkaline phosphatase, nitroblue tetrazolium (NBT) chloride (50 mg, Sigma) was dissolved in 70 percent dimethylformamide.
  • NBT nitroblue tetrazolium
  • FAID14 is a murine anti-idiotypic antibody (lgG2a, K isotype) deposited as ATCC No. CRL 10256 directed against CC49.
  • FAID14 was purified using a Nygene Protein A affinity column (Yonkers, NY). The manufacturer's protocol was followed, except that 0.1 M sodium citrate, pH 3.0 was used as the elution buffer. Fractions were neutralized to pH-7 using 1.0 M Tris-HCI pH 9.0. The biotinylation reaction was set up as follows. FAID14 (1 mg, 100 ⁇ L in water) was mixed with 100 ⁇ L of 0.1 M Na 2 CO 3 pH 9.6.
  • Biotinyl- ⁇ -amino-caproic acid N-hydroxy succinimide ester (Biotin-X-NHS) (Cal biochem, LaJolla, CA) (2.5 mg) was dissolved in 0.5 mL dimethylsulfoxide. Biotin-X-NHS solution (20 ⁇ L) was added to the FAID14 solution and allowed to react at 22°C for 4 hours. Excess biotin and impurities were removed by gel filtration, using a Pharmacia Superose 12 HR10/30 column (Piscataway, NJ). At a flow rate of 0.8 mL/min, the biotinylated FAID14 emerged with a peak at 16.8 min. The fractions making up this peak were pooled and stored at 4°C and used to detect the CC49 idiotype as determined by the CC49 V L and V H CDRs.
  • Isoelectric points were predicted using a computer program called PROTEIN-TITRATE, available through DNASTAR (Madison, WI). Based on amino acid composition with an input sequence, a MW value is given, in addition to the pl. Since Cys residues contribute to the charge, the count was adjusted to 0 for Cys, since they are all involved in disulfide bonds.
  • pl's were determined using Isogel agarose IEF plates, pH range 3-10 (FMC Bioproducts, Rockland, ME).
  • a Biorad Bio-phoresis horizontal electrophoresis cell was used to run the IEF, following the directions of both manufacturers.
  • the electrophoresis conditions were : 500 volts (limiting), at 20 mA current and 10 W of constant power. Focusing was complete in 90 min.
  • IEF standards were purchased from Biorad; the kit included phycocyanin, ⁇ -lactoglobulin B, bovine carbonic anhydrase, human carbonic anhydrase, equine myoglobin, human hemoglobins A and C, 3 lentil lectins and cytochrome C, with pl values of 4.65, 5.10, 6.00, 6.50, 7.00, 7.10 and 7.50, 7.80, 8.00, and 8.20 and 9.60, respectively. Gels were stained and destained according to the directions provided by FMC.
  • HPLC high performance liquid chromatography
  • PCR polymerase chain reactions
  • a reaction mixture consisting of: 150 picograms (pg) plasmid target (pSCFVUHM); 100 pmoles primers; 1 ⁇ L Perkin-Elmer-Cetus (PEC, Norwalk, CT) Ampli-Taq polymerase; 16 ⁇ L of 10 mM dNTPs and 10 ⁇ L of 10X buffer both supplied in the PEC kit; and sufficient water to bring the volume to total volume to 100 ⁇ L.
  • the PCR reactions were carried out essentially as described by the manufacturer.
  • Reactions were done in a PEC 9600 thermocycler with 30 cycles of: denaturation of the DNA at 94°C for 20 to 45 sec, annealing from between 52 to 60°C for 0.5 to 1.5 min., and elongation at 72°C for 0.5 to 2.0 min.
  • Oligonucleotide primers were synthesized on an Applied Biosystems (Foster City, CA) 380A or 391 DNA synthesizer and purified as above.
  • Ligation reactions using 100 ng of vector DNA and a corresponding 1 : 1 stoichiometric equivalent of insert DNA were performed using a Stratagene (La Jolla, CA) T4 DNA ligase kit following the manufacturer's directions. Ligation reactions (20 ⁇ L total volume) were initially incubated at 18°C and allowed to cool gradually overnight to 4°C.
  • Transformations were performed utilizing 100 ⁇ L of Stratagene E. coli AG 1 competent cell (Stratagene, La Jolla, CA) according to the directions provided by the manufacturer. DNA from the ligation reactions (1-5 ⁇ L) were used. After the transformation step, cells were allowed to recover for 1 hr in Luria broth (LB) at 37°C with continuous mixing and subsequently plated onto either 20 ⁇ g/mL chloramphenicol containing (CAM 20) Luria agar for pSCFVUHM, p49LHLH or p49LHHL or 100 ⁇ g/mL ampicillin (AMP 100) Luria agar plates (LB-AMP 100) for clones contai ning the plasmid pSL301 or subsequent constructions derived from pSL301.
  • LB Luria broth
  • AMP 100 ampicillin
  • Bacterial plasmids were isolated from LB broth culture containing the appropriate drug to maintain selection pressure using Promega (Madison, WI) Magic mini-prep plasmid preparation kits. The kit was used per the manufacturer's specifications.
  • p49LHLH and p49LHHL Two plasmids, designated p49LHLH and p49LHHL, were constructed to produce multivalent single chain antibodies.
  • the host cell containing p49LHLH produced a polypeptide which can be designated by V L -L-V H -L-V L -L-V H where V L and V H are the light and heavy cahin variable regions of CC49 antibody and linker (L) is a 25 amino acid linker having the sequence (SEQ ID NO: 5).
  • the host cell containing p49LHHL produced a polypeptide which can be designated by V L -L-V H -L-V H -L-V L where V L and V H are the light and heavy chain variable domains of the CC49 antibody and L is a peptide linker having the amino acid sequence indicated above.
  • nucleotide sequence (SEQ ID NO: 6) and amino acid sequence (SEQ ID NO: 7) of the CC49 V L -L-V H -L-V L -L-V H (p49LHLH) are given in Figure 6.
  • nucleotide sequence (SEQ ID NO: 8) and amino acid sequence (SEQ ID NO: 9) of the CC49 V L -L-V H -L-V H -L-V L (p49LHHL) are given in Figure 7.
  • pSL301 HT The construction of pSL301 HT is illustrated in Figure 8.
  • the Bacillus lichiformis penicillinase P (penP) terminator sequence was removed from the plasmid designated pSCFV UHM by a 45 minute digest with Nhe I and BamH I, excised from a 4.5 percent polyacrylamide gel after electrophoresis, electroeluted, ethanol precipitated and ligated into the same sites in the similarly prepared vector: pSL301 (Invitrogen, San Diego, CA).
  • a procedure for preparing pSCFV UHM is given is U.S. patent application Ser. No. 07/935,695 filed August 21, 1992, the disclosure of which is hereby incorporated by reference.
  • pSCFV UHM contains a nucleotide sequence for a penP promoter; a unique Nco 11 restriction site; CC49 V L region; Hind III restriction site; a 25 amino acid linker; a unique a Xho I restriction site; CC49 V H region; Nhe I restriction site; penP terminator; and BamH I restriction site (see, Figure 8).
  • the penP promoter and terminator are described in Mezes, et al. (1983), J. Biol. Chem., 258 11211-11218 (1983).
  • the V H sequence was made by PCR with oligos 5' SCP1 and 3'oligo SCPS using pSCFV U HM as the target for PCR amplification.
  • the DNA sequence for SCP1 (SEQ ID NO: 10) and SCPS (SEQ ID NO: 11) are as follows: The underlined portion indicates the endonuclease restriction sites.
  • the amplified V H DNA was purified from a 4 percent PAG, electroeluted ethanol precipitated and dissolved in 20 ⁇ L water.
  • the V H sequence was digested with Xho I and Nhe I restriction enzymes and used as the insert with the pSL301 T vector which had been digested with the same restriction enzymes and subsequently purified.
  • a standard ligation reaction was done and an aliquot (4 ⁇ L) used to transform competent E. coli AG1 cells.
  • the transformed cells were plated onto LB AMP100 agar plates. Candidate dones were picked from a Nhe I and Xho I digest screen that revealed that the CC49V H insert had been obtained.
  • DNA sequencing was performed to verify the sequence of the CC49V H with United States Biochemical (USB) (Cleveland, Ohio) Sequence kit and sequencing primers pSL301SEQB (a 21 bp sequencing primer which annealed in the pSL301 vector 57 bp upstream from the Xho I site) and CC49VHP, revealed clones with the correct CC49V H sequence in pSL301HT.
  • This plasmid was used as the starting point in the construction of both pSL301-HHLT and pSL301-HLHT. The sequencing oligos used are shown here.
  • pSL301 HT 5 ⁇ g
  • SCP6B 5' oligo
  • SCPS 3' oligo
  • the nucleotide sequence (SEQ ID NO: 14) of SCP6B is as follows:
  • the oligo SCP6B also contains part of the coding region for the linker (bp 8-76 of SEQ ID NO: 14).
  • the portion of the oligo designed to anneal with the CC49VH target in pSCFV UHM is from bp77-90 in SEQ ID NO: 14.
  • the underlined sequence corresponds to the Fsp I site.
  • the resulting PCR insert was purified, digested with Fsp I and Nhe I and used in a ligation reaction with the pSL301 HT Eco47 III-Nhe I vector ( Figure 7). Competent E. coli AG1 cells were used for the transformation of this ligation reaction (3 ⁇ L) and were plated on LB-AMP100 agar plates. Two clones having the correct size Xho I-Nhe I insert representative of the pSL301 HHT product were sequenced with the oligo SQP1 and a single clone with the correct sequence (nucleotides 1124-1543 of Figure 7) was chosen for further construction.
  • the nucleotide sequence of SQP1 (SEQ ID NO: 15 is as follows:
  • the final linker-V L subunit (bp 1544-1963, Figure 7) was generated using the 5'oligo, SCP7b and the 3' oligo, SCP8a, using pSCFV UHM as the target for the PCR.
  • the nucleotide sequence of SCP7b (SEQ ID NO: 16) is as follows: The underlined nucleotides correspond to an Fsp I site.
  • the nucleotide sequence of SCP8a (SEQ ID NO: 17) is as follows:
  • the first set of underlined nucleotides correspond to an Nhe I site, while the other corresponds to an Afl II site.
  • Nucleotides B-76 of SCP70 code for the linker (nucleotides 1544-1612 of Figure 7) while nucleotides 77-99 which anneal to the V L correspond to 1613-1635 of Figure 7.
  • the primer SCP8a has a short tail at its 5' end, a Nhe I restriction site, a stop codon, an Afl II restriction site and the last 21 bases of the V L .
  • the plasmid pSL301HHLT was digested with Xho I and Nhe I, purified, and the resulting 1179 bp V H -linker-V H -linker-V L segment ligated into pSCFV UHM, which had been cut with the same restriction enzymes and the larger vector fragment purified, to form p49LHHL.
  • the ligation reaction (4 ⁇ L aliquot) was used to transform competent E. coli AG 1 cells (Stratagene) and plated onto LBCAM20 agar plates. A single clone which had a plasmid with the correct restriction enzyme map was selected to contain p49LHHL.
  • the p49LHHL contains a penP promoter and a nucleotide sequence for the CC49 multivalent single chain anti body scFv2: V L -L-V H -L-V H -L-V L or CC49 scFv2 (LHHL).
  • FIG. 10 The construction of p49LHLH is schematically represented in Figure 10.
  • a linker-V L subunit was generated with the 5' oligo SCP7b and the 3'oligo SCP9 (SEQ ID NO: 19).
  • the SCP7b oligo(nucleotides 8-76) codes for the linker in Figure 6 (corresponding to nucleotides 1124-1192) and annealed to the pSCFV UHM target for the PCR (nucleotides 77-99) corresponding to nucleotides 1193-1215 of the V L in Figure 6.
  • SCP9 has a Nhe I site (first underlined nucleotides) and an Eco47 III site (second underlined nucleotides) which are restriction sites needed for making the pSL301HLT ready to accept the next V domain.
  • Nucleotides 18-23 of SCP9 correspond to nucleotides 1532-1537 of Figure 6 (coding for the first 2 amino acids of the linker).
  • nucleotides 24-46 correspond to nucleotides 1508-1531 of Figure 6 which was also the annealing region for SCP9 in the PCR.
  • the plasmid pSL301 HT was digested with Eco47 III and Nhe I and the larger vector fragment was purified for ligation with the linker-CC49V L DNA insert fragment from the PCR which had been treated with Fsp I and Nhe I and purified.
  • the ligation mixture (3 ⁇ L) was used to transform E. coli AG 1 competent cells and one colony having the correct Xho I-Nhe I size fragment was sequenced using the oligo PENPTSEQ2.
  • the nudeotide sequence (SEQ. ID NO.20) is as follows:
  • SCP6c corresponds to an Eco47 III site.
  • SCP6C was used as the 5' oligo, with SCP10 as the 3' oligo in a PCR to generate a linker CC49 V L segment.
  • the nudeotide sequence (SEQ ID NO: 22) is as follows:
  • the underlined sequence in SCP10 corresponds to the Nhe I site found at nucleotides 1958-1963 in Figure 6.
  • the PCR insert was digested this time only with Nhe I and purified.
  • the vector (pSL301 HLT) was digested at the Eco47 III site (that had been formed) and Nhe I and purified.
  • the insert and vector were ligated and an aliquot (3 ⁇ L) used to transform competent E. coli AG1 cells. This was plated on LB-AMP100 plates and candidate clones screened with Xho I and Nhe I. Three clones having the correct size DNA were obtained. Two of these clones were sequenced using the oligo 49VLCDR3(+) and SQP1.
  • the nucleotide sequence (SEQ ID NO:23) of 49VLCDR3(+) is as follows:
  • p49LHLH for expression in E. coli pSL301 HLHT (5 ⁇ g) was digested with Nhe I and Xho I, and the smaller insert fragment containing the V H -L-V L -L-V H sequence purified. It was ligated with the larger purified vector fragment from a digest of pSCFV UHM (5 ⁇ g) with Xho I and Nhe I. An aliquot of the ligation mix (4 ⁇ L) was used to transform competent E. coli AG1 cells. The transformation mix was plated on LB-CAM20 plates, and a representative clone for p49 LHLH was selected on the basis of a correct restriction enzyme map (see Figure 10) and biological activity toward TAG-72.
  • E. coli periplasmic fractions were prepared from 1.0 L overnight cultures of both p49LHLH and p49LHHL. Briefly, the culture was divided into 4 X 250 mL portions and centrifuged at 5,000 rpm for 10 minutes in a Sorvall GS-3 rotor. The pelleted cells were washed and resuspended in 100 mL each of 10 mM Tris-HCl pH 7.3 containing 30 mM NaCI. The cells were again pelleted and washed with a total of 100 mL 30 mM Tris-HCI pH 7.3 and pooled into one tube.
  • scFv2 E. coli periplasmic fractions were prepared from 1.0 L overnight cultures of both p49LHLH and p49LHHL. Briefly, the culture was divided into 4 X 250 mL portions and centrifuged at 5,000 rpm for 10 minutes in a Sorvall GS-3 rotor. The pelleted cells were
  • coli periplasmic fraction was clarified further by filtration through a 0.2 ⁇ m Nalge (Rochester, NY) filter apparatus and concentrated in Amicon (Danvers, MA) Centriprep 30 and Centricon 30 devices to a volume of less than 1.0 mL.
  • the concentrated peri plasmic shockates from either the p49LHLH or p49LHHL clones were injected onto a Pharmacia (Piscataway, NJ) Superdex 75 HR 10/30 HPLC column that had been equilibrated with PBS. At a flow rate of 0.5 mL/minute, the product of interest, as determined by competition ELISA, had emerged between 21 through 24 minutes.
  • the active fractions were pooled, concentrated as before and dialyzed overnight using a system 500 Microdialyzer Unit (Pierce Chemical) against 20 mM Tris-HCI pH 7.6 with 3-4 changes of buffer and using an 8,000 MW cutoff membrane. The sample was injected on a Pharmacia Mono Q HR 5/5 anion exchange HPLC column.
  • the active fractions were in each case concentrated, dialysed against 50 mM MES pH 5.8 overnight and injected on a Pharmacia Mono S HR 5/5 cation exchange column.
  • the two fractions of interest from this purification step as determined by SDS-PAGE and ELISA, fractions 5 and 6, eluted just before the start of the gradient, so they had not actually bound to the column. Fractions 5 and 6 were consequently pooled for future purification.
  • the isoelectric points (pl) of the constructs was predicted using the DNASTAR (Madison, WI) computer program Protein-titrate. Based on amino acid composition, a MW and pl value was calculated.
  • pls were determined using FMC Bioproducts (Rockland, ME) Isogel IEF plates, pH range 3-10.
  • a Biorad (Richmond, CA) electrophoresis unit was used to run the IEF, following the directions of both manufacturers.
  • the electrophoresis conditions were as follows: 500 V (limiting) at 20 mA and at 10 W of constant power. Focusing was complete in 90 minutes.
  • CC49 antibodies such as the IgG, scFv2 (LHLH and LHHL) were quantitated by measuring the absorbence spectrophotometrically at 280 nm. Molar absorbtivity values, ⁇ M , were determined for each using the formula cited above by Wetlaufer.
  • the E 0.1% (280 nanometers) values for CC49 IgG, CC49 scFv2 LHLH, CC49 scFv2 LHHL and CC49 scfv were 1.49, 1.65, 1.65 and 1.71, respectively.
  • Relative activities of the CC49 scFv2 species LHLH and LHHL were compared with the IgG and a monomer scfv form with a FLAG peptide at the COOH terminus.
  • Percent competition was determined from the ELISA data by the following equation: Zero competition - sample reading (OD405-450 nm) zero competition - 100 percent competition x 100
  • the "zero competition" value was determined by mixing (1 : 1) one percent BSA with the biotinylated CC49 (3 X 10-14 moles) while the 100 percent competition value was based on a 5 ⁇ g/mL sample of CC49 IgG mixed with the biotinylated CC49 IgG.
  • the data are presented in Figure 11. Absorbence values for the samples were measured at 405 nm - 450 nm. The average of triplicate readings was used. Initially samples (25 pL) were applied to the TAG-72 coated microliter plates at 1.0 X 10-10 moles of binding sites/mL.
  • Biotinylated CC49 (4 ⁇ g/ ⁇ L diluted 1:20,000 - used 25 ⁇ L) diluted the samples by a factor of 2. Serial dilutions (1:2) were performed. Both forms of the scFv2 are approximately equivalent to the IgG (see Figure 11).
  • a CC49 scFv monomer was compared to a Fab fragment, both of which are monovalent and these were also shown to be equivalent in their binding affinity for TAG-72.

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Claims (10)

  1. Multivalenter einzelkettiger Antikörper, welcher zwei oder mehr einzelkettige Antikörperfragmente umfaßt, wobei jedes einzelkettige Antikörperfragment an ein Antigen spezifisch bindet, worin die einzelkettigen Antikörperfragmente durch einen ersten Peptidlinker kovalent verknüpft sind, welcher eine Aminosäuresequenz von
    Figure 00450001
    enthält, und jedes einzelkettige Antikörperfragment
    (a) ein erstes Polypeptid, umfassend eine variable Domäne einer leichten Kette;
    (b) ein zweites Polypeptid, umfassend eine variable Domäne einer schweren Kette; und
    (c) einen zweiten Peptidlinker, welcher das erste und das zweite Polypeptid zu einer funktionellen Bindungsgruppe verknüpft,
    umfaßt.
  2. Multivalenter einzelkettiger Antikörper nach Anspruch 1, worin die variable Region der leichten Kette und die variable Region der schweren Kette aus Antikörpern gegen Tumor-assoziiertes Glykoprotein 72-Antigen (TAG-72) erhalten sind.
  3. Multivalenter einzelkettiger Antikörper nach Anspruch 1, worin die variable Region der leichten Kette eine Aminosäuresequenz von
    Figure 00450002
    und die variable Region der schweren Kette eine Aminosäuresequenz von
    Figure 00460001
    aufweist.
  4. Multivalenter einzelkettiger Antikörper nach Anspruch 1, worin der erste Peptidlinker eine Aminosäuresequenz mit 25 bis 30 Aminosäureresten aufweist.
  5. Multivalenter einzelkettiger Antikörper nach Anspruch 1, worin der zweite Peptidlinker eine Aminosäuresequenz mit von 10 bis 30 Aminosäureresten aufweist.
  6. Multivalenter einzelkettiger Antikörper nach Anspruch 1, worin der erste und der zweite Peptidlinker im wesentlichen dieselbe Aminosäuresequenz aufweisen.
  7. Multivalenter einzelkettiger Antikörper nach Anspruch 6, worin der zweite Peptidlinker eine Aminosäuresequenz aufweist, die mit derjenigen des ersten Peptidlinkers identisch ist.
  8. DNA-Sequenz, welche für einen multivalenten einzelkettigen Antikörper codiert, wobei der multivalente einzelkettige Antikörper zwei oder mehr einzelkettige Antikörperfragmente umfaßt, wobei jedes Fragment eine Affinität für ein Antigen besitzt, worin die Fragmente durch einen ersten Polypeptidlinker kovalent verknüpft sind, welcher eine Aminosäuresequenz von
    Figure 00460002
    enthält,
    und jedes Fragment
    (a) ein erstes Polypeptid, umfassend eine variable Domäne einer leichten Kette;
    (b) ein zweites Polypeptid, umfassend eine variable Domäne einer schweren Kette; und
    (c) einen zweiten Peptidlinker, welcher das erste und das zweite Polypeptid zu einer funktionellen Bindungsgruppe verknüpft,
    umfaßt.
  9. DNA-Sequenz nach Anspruch 8, welche für einen multivalenten einzelkettigen Antikörper codiert, worin die variable Region der leichten Kette und die variable Region der schweren Kette aus Antikörpern gegen Tumor-assoziiertes Glykoprotein 72-Antigen (TAG-72) erhalten sind.
  10. DNA-Sequenz nach Anspruch 8, worin die für das erste Polypeptid codierende Sequenz im wesentlichen homolog zu der Sequenz:
    Figure 00470001
    ist und das erste Polypeptid die Eigenschaft der funktionellen Bindung an TAG-72 beibehält, und worin die für das zweite Polypeptid codierende Sequenz im wesentlichen homolog zu der Sequenz
    Figure 00470002
    ist und das zweite Polypeptid die Eigenschaft einer funktionellen Bindung an TAG-72 beibehält.
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US5877291A (en) 1999-03-02
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